Agricultural output has risen dramatically during the last half of the twentieth century. A large portion of this increase has been attributed to the development and use of hybrid seed varieties in core crops such as corn, sorghum, sunflower, alfalfa, canola and wheat. The success of hybrid seed varieties is due to a phenomenon called heterosis, where hybrid plants display a more desirable phenotype than either of the two inbred parental lines used to produce the hybrid plant. Heterosis has been observed in a number of plant traits including yield, plant height, biomass, resistance to disease and insects, tolerance to stress and others. These heterotic traits are polygenic in nature, resulting in their characteristic range of phenotypes, rather than traditional discrete Mendelian phenotypes. The polygenic nature of the traits results in complex patterns of inheritance such that the underlying components for the observed heterotic phenotypes is still a matter of debate in the plant science community.
Because of the economic value of heterosis, there have been several attempts to use molecular biology techniques to augment traditional hybrid plant breeding programs. The bulk of the efforts have focused on either mRNA (messenger RNA) or genomic DNA. The mRNA approach is extremely difficult as comparisons require tissue samples selected from the same portion of the plant, at the same developmental time, and in the same or highly similar environmental conditions. The process is further complicated as a researcher needs to determine which plant portion or developmental stage will yield the best results for predicting the degree of a particular heterotic phenotype of interest. As a result of these complications, mRNA-based predictions frequently have high levels of noise and have low accuracy in the prediction of the degree of a heterotic phenotype.
The use of genomic DNA to predict the degree of one or more heterotic phenotypes has been similarly disappointing. Initial efforts used subtractive hybridization or fluorescent in situ hybridization in order to identify copy number differences in inbred plant lines. These techniques do not produce easily quantifiable results and can only detect gross differences in copy numbers, such as a doubling or complete elimination. This is a significant problem in polyploid plants as chromosomal duplications and other evolutionary events have resulted in genes with multiple copies, some of which are pseudogenes, throughout the plant genome. These higher copy numbers greatly reduce the usefulness of the genomic DNA approaches as they are unable to accurately detect the addition or deletion of a single copy of a gene represented three or more times in the genome.
Another genomic approach has been the use of genetic markers to predict heterosis. In these techniques, RFLP markers as well as other traditional markers have been used. Researchers have attempted to use genetic markers to predict the degree of a heterotic phenotype with some success, so long as the potential parent plants belong to the same heterotic groups that were used in the initial crosses to generate the correlational data upon which the prediction is based. Once plants from other heterotic groups are used, the heterotic phenotype predictive ability of genetic markers greatly diminishes. The reason for the loss of predictive ability has been attributed to insufficient linkage of the markers to quantitative trait loci controlling the trait of interest, and a lack of gametic phase linkage disequilibrium between the marker and quantitative trait loci alleles. This diminished predictive ability severely limits the use of genetic markers in plant breeding programs.
Based on these efforts, the application of molecular biology techniques to the prediction of the degree of a heterotic phenotype has been problematic at best. Despite years of research, there has yet to be a satisfactory method developed.
Comparative Genome Hybridization (CGH) is a technique that has been employed to study chromosomal abnormalities in animal cells. A major area of CGH use has been in analyzing cancer mutations in an effort to better identify cancer cells in order to select more effective courses of therapy. CGH is particularly effective in animal cells as there are typically two copies of any given gene in the genome (one from each parent). Additionally, entire genomes for mammals are currently known. Researchers have been able to take advantage of the low duplication and genome sequence information to identify duplicated and deleted chromosomal regions. This information can then be used to identify the changes that have transformed normal cells into cancerous cells. However, the complete genome sequence of several major crops is not known at present. As a result, there has been little use of CGH in plants and doing so requires overcoming the numerous differences that arise when working with plant genomics.